Solution and Membrane-Bound Conformations of the Tandem C2A and C2B Domains of Synaptotagmin 1: Evidence for Bilayer Bridging |
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Authors: | Dawn Z Herrick Hao Huang Jeffrey F Ellena |
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Institution: | 1 Department of Chemistry and Biophysics Program at the University of Virginia, Charlottesville, VA 22904-4319, USA 2 Division of Computational Bioscience and Center for Information Technology, National Institutes of Health, Bethesda, MD 20892, USA |
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Abstract: | Synaptotagmin 1 (syt1) is a synaptic vesicle membrane protein that functions as the Ca2+ sensor in neuronal exocytosis. Here, site-directed spin labeling was used to generate models for the solution and membrane-bound structures of a soluble fragment of syt1 containing its two C2 domains, C2A and C2B. In solution, distance restraints between the two C2 domains of syt1 were measured using double electron-electron resonance and used in a simulated annealing routine to generate models for the structure of the tandem C2A-C2B fragment. The data indicate that the two C2 domains are flexibly linked and do not interact with each other in solution, with or without Ca2+. However, the favored orientation is one where the Ca2+-binding loops are oriented in opposite directions. A similar approach was taken for membrane-associated C2A-C2B, combining both distances and bilayer depth restraints with simulated annealing. The restraints can only be satisfied if the Ca2+ and membrane-binding surfaces of the domains are oriented in opposite directions so that C2A and C2B are docked to opposing bilayers. The result suggests that syt1 functions to bridge across the vesicle and plasma membrane surfaces in a Ca2+-dependent manner. |
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Keywords: | DEER double electron-electron resonance EGTA ethylene glycol bis(β-aminoethyl ether)N N&prime -tetraacetic acid MTSL (1-oxyl-2 2 5 5-tetramethylpyrroline-3-methyl)methanethiosulfonate PDB Protein Data Bank POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine POPS 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine R1 and R7 spin-labeled side chains produced by derivatization of a cysteine with a methanethiosulfonate spin label SNARE soluble N-ethylmaleimide-sensitive factor attachment protein receptor syt1 synaptotagmin 1 syt1C2AB soluble fragment of syt1 containing the C2A and C2B domains |
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